Need help on MR-VP test

In article <01HNUX051MGI0027IO at vax.rhodes.edu> HILL at NS.RHODES.EDU writes:
>From: HILL at NS.RHODES.EDU>Subject: Re: Need help on MR-VP test
>Date: 7 Mar 1995 12:56:41 -0800
>Pong Nim writes:
>>I am having problem obtaining a positive test for Methy Red (MR)
>>and Voges-Proskauer (VP) reaction.
>>I have grew Enterobacter aerogenes & Serratia marcescens in
>>MR-VP broth at 35 C for 48 hrs. Five drops of MR were added.
>>on one set of MR-VP broth. Twelve drops of VP reagent I and
>>three drops of VP reagent II were added to the second set of
>>MR-VP broth. All reagents were made fresh on the day of the test.
>>Still I can't seem to get any positive reaction at all. What
>>am I doing wrong. Your helpful advice would greatly be appreciate.
>In reply:
>The Enterobacter, of course, should be negative for the MR test -- and while
>some S. marcescens will be positive, not all will be. So probably you just
>have an MR-negative strain of Serratia. Try over with E. coli, and I bet it'll
>work.
>As for the V.P., often that needs to go for longer than 2 days -- typical
>protocols may specify *5* days. Try both of your strains after a longer
>incubation. Also, be sure to put parafilm over the tube and shake it
>vigorously several times over several minutes, before deciding it's negative.
>The reaction needs oxygen. (Don't expect necessarily an instant result - it
>might take as long as a half an hour. I've also seen some suggestions that the
>mixture be heated gently, but I've never had to do that.) Finally, the volumes
>you mention *might* be at fault - you don't say what exact volumes you're using
>- I've had good results with a half ml of each reagent for every ml of medium.
The above is excellent advice. I test over several days; remove a sub sample
of 10 drops, add 6 drops of alpha-naphthol solution (always first) and 2 drops
of 40% KOH. Mix and leave on a slant for up to 30 min. Sloping the tube
increases the air interface and hence oxygen transfer. Don't read the test
after 30 minutes; a coppery colour can develop which might tempt one to think
of a weak positive. Have a look at MacFaddin J.F., Biochemical tests for
identification of medical bacteria 2 edit. 1980, Williams & Wilkins. Contains
all you need to know and more about many identification tests for bacteria.
Check to make sure that glucose has been added to the medium and that it has
not been degraded by excessive, prolonged heating during autoclaving. Consider
using the Coblenz KOHsolution which in addition to the 40% KOH also contains
0.3% creatine.